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doi:10.2204/iodp.proc.347.109.2015

Microbiology

Hole M0065C was drilled specifically for microbiology, interstitial water chemistry, and unstable geochemical parameters at Site M0065. Counts of microbial cells were made by fluorescence microscopy using the acridine orange direct count (AODC) method and by flow cytometry (FCM) using SYBR green DNA stain during the OSP. Further counts by fluorescence microscopy will be conducted after the OSP using both acridine orange and SYBR green staining.

A total of 15 sediment samples were counted for microbial cell numbers on the ship and during the OSP (Table T10). Of these, 11 samples were enumerated using the flow cytometer, and all 15 samples were enumerated using the epifluorescence microscope counting technique.

The most striking observations for these data are that cell counts by AODC are extremely high in Unit I and that the two cell counting techniques do not produce similar results in the upper half of this hole (Fig. F19; see “Microbiology” in the “Site M0063” chapter for a similar pattern of results [Andrén et al., 2015b]). The uppermost cell count at 3.53 mbsf, by FCM, was 9.42 × 10⁸ cells/cm³, whereas 1.23 × 10¹⁰ cells/cm³ were determined by AODC, a 13-fold difference. In the lower half of this hole, data from both counting techniques appeared similar. The minimum microbial populations were determined as 7.10 × 10⁷ cells/cm³ at 23.33 mbsf by FCM and 6.18 × 10⁷ cells/cm³ at 13.43 mbsf by AODC.

Regression analyses of both data profiles indicated different trends shallower and deeper than ~12 mbsf (Fig. F19). The decrease in cell numbers with depth was significantly steeper shallower than this depth compared to deeper than this depth both for FCM (F = 8.67; degree of freedom [df] = 1.7; P < 0.025) and for AODC (F = 45.82; df = 1.11; P << 0.001). In the upper 12 m the change in cell numbers with depth was approximately 2.3 times steeper for AODC compared to FCM. This compares to a difference of 2.4 times steeper in Hole M0063E (see “Microbiology” in the “Site M0063” chapter [Andrén et al., 2015b]). Deeper than 12 mbsf, regression lines from the two depth profiles were not significantly different from each other (F = 0.577; df = 1.11 [not significant]), which was confirmed by a paired sample t-test (t = 0.598; df = 6 [not significant]).

This is the same situation that was encountered in samples from Hole M0063E where in the upper part of the hole many of the cells were aggregated in clumps of “fluff,” making accurate enumeration by either method difficult or impossible (see interpretation in “Microbiology” in the “Site M0063” chapter [Andrén et al., 2015b]), and this resulted in significant underestimation of cell numbers by FCM compared to AODC. An improved processing technique will need to be developed to deal with these samples, and it is possible that even higher concentrations of cells will be detected than already reported for this hole. The fluff is possibly bacterially derived exopolysaccharide causing cell clumping, and it is of note that this depth interval of difficult-to-count samples coincides with both the largest differences between the two counting techniques and the presence of organic-rich sediment (see “Geochemistry”; Figs. F13A, F19).

The profile break at 12 mbsf does seem to be related to sediment column stratigraphy, as this depth roughly correlates with the transition between the upper brackish-marine sediments and the lower late glacial lake clay deposits at the Unit II/III boundary. In the organic-rich sediments shallower than 12 mbsf, bacterial degradation of organic matter has produced a broad maximum in alkalinity that reaches 38 meq/L in the upper part of the hole compared to 4–5 meq/L in the lower half of the hole (see “Geochemistry”). The high cell numbers thus coincide with the interval of high degradation rate of organic matter. Conversely, salinity decreases more or less linearly from the sediment surface to ~30 mbsf and does not appear related to the cell profiles (Fig. F19).

As was reported for Sites M0059, M0060, M0061, and M0063, cell numbers were very high and, with one exception, all cell counts exceeded the upper prediction limit for the global regression (Fig. F19). The maximum deviation from the global regression was at 29.93 mbsf for the FCM profile with a 36-fold higher cell number, whereas for the AODC technique the greatest deviation was at 6.83 mbsf with a 370-fold higher number. It is of note that the cell density of 1.23 × 10¹⁰ cells/cm³ at 3.53 mbsf is among the highest observed in all marine sediments examined by scientific drilling.

When the data from both techniques are plotted against each other, results from deeper than 12 mbsf cluster around the line of x = y (Fig. F20). Data are too clustered for a regression line to be calculated and compared to the x = y line. Results from shallower than 12 mbsf clearly deviate from the x = y line with higher numbers obtained by the AODC method.

Perfluorocarbon (PFC) tracer was detected in the liner fluid and exteriors of all cores, indicating continuous PFC delivery into the microbiology borehole (Table T11). Liner fluid PFC concentrations varied over 1–2 orders of magnitude (Fig. F21A), indicating variations in the rates of PFC delivery and mixing into the drilling fluid stream. Most of the measured PFC concentrations were below the target concentration of 1 mg PFC/L. Nonetheless, the liner fluid PFC concentrations are on average considerably higher than at the other three sites, remaining above 10^–5 g PFC/L in all samples (apart from Core 2H) and in two cores (347-M0065C-3H and 4H) closely approaching the PFC target concentration. PFC was above detection in all core halfway samples and all but two interior samples (Cores 6H and 7H; Fig. F21B). As at Sites M0059 and M0060, contamination was relatively highest in the uppermost part of the hole (Core 347-M0065C-2H) and showed no depth- or lithology-related trend below (Figs. F19, F21C). Remarkably, PFC contamination was 2–3 orders of magnitude higher in several “core halfway” sections than core exteriors (Cores 7H, 10H, and 12H), a phenomenon that was only observed once across the three other microbiology sites (Core 347-M0063E-23H).

Compared to the other sites, Site M0065 has a higher fraction of cores that are suitable for microbiological analyses. No contamination could be detected in interiors of Cores 347-M0065C-6H and 7H. Moreover, Core 3H only showed marginal evidence of contamination, despite having the highest PFC concentration in the liner fluid of all cores, and Cores 9H, 10H, and 12H were calculated to have potentially fewer than 100 contaminant cells/cm³ in core interiors (Table T11; Fig. F21D).

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